The Journal of Membrane Biology

, Volume 246, Issue 1, pp 7–11 | Cite as

Characterization of Two Mammalian Cortical Collecting Duct Cell Lines with Hopping Probe Ion Conductance Microscopy

  • Xuewei Chen
  • Hui Zhu
  • Xiao Liu
  • Hujie Lu
  • Ying Li
  • Jing Wang
  • Hongtao Liu
  • Jianning Zhang
  • Qiang Ma
  • Yanjun Zhang


We morphologically and physiologically characterized Madin–Darby canine kidney (MDCK) cell and mouse principal cell of kidney in cortical collecting duct (mpkCCD) via hopping probe ion conductance microscopy, transepithelial electrical resistance (TEER) measurements, and single-channel recordings. The specific membrane structures of microvilli and tight junctions were clearly observed in MDCK and mpkCCD cell monolayers. The electrophysiological functions of epithelial Na+ channel in MDCK and mpkCCD cells were further characterized by measuring amiloride-sensitive TEER values for the whole-cell monolayer and detecting the ion channel activities with patch clamping. Our results provide more morphological and functional information to help better utilize these two mammalian CCD cell lines for mechanism studies of sodium absorption and reabsorption in the distal nephron.





This work was supported by the National Natural Science Foundation of China (no. 30971184), International Science & Technology Cooperation Program of China (no. 2011DFG33430), National Basic Research Program of China (973 Program) (no. 2011CB933100).


  1. Bubien JK (2010) Epithelial Na+ channel (ENaC), hormones, and hypertension. J Biol Chem 285:23527–23531PubMedCrossRefGoogle Scholar
  2. Conceicao N, Henriques NM, Ohresser MCP, Hublitz P, Schule R, Cancela ML (2003) Molecular cloning of the matrix Gla protein gene from Xenopus laevis. Eur J Biochem 269:1947–1956CrossRefGoogle Scholar
  3. Dooley R, Harvey BJ, Thomas W (2012) Non-genomic actions of aldosterone: from receptors and signals to membrane targets. Mol Cell Endocrinol 350:223–234PubMedCrossRefGoogle Scholar
  4. Gorelik J (2005) Aldosterone acts via an ATP autocrine/paracrine system: the Edelman ATP hypothesis revisited. Proc Natl Acad Sci USA 102:15000–15005PubMedCrossRefGoogle Scholar
  5. Gorelik J, Zhang Y, Shevchuk AI, Frolenkov GI, Sánchez D, Lab MJ, Vodyanoy I, Edwards CRW, Klenerman D, Korchev YE (2004) The use of scanning ion conductance microscopy to image A6 cells. Mol Cell Endocrinol 217:101–108PubMedCrossRefGoogle Scholar
  6. Grossmann C, Gekle M (2009) New aspects of rapid aldosterone signaling. Mol Cell Endocrinol 308:53–62PubMedCrossRefGoogle Scholar
  7. Hoh JH, Schoenenberger CA (1994) Surface morphology and mechanical properties of MDCK monolayers by atomic force microscopy. J Cell Sci 107(pt 5):1105–1114PubMedGoogle Scholar
  8. Ishikawa T, Marunaka Y, Rotin D (1998) Electrophysiological characterization of the rat epithelial Na+ channel (rENaC) expressed in MDCK cells. Effects of Na+ and Ca2+. J Gen Physiol 111:825–846PubMedCrossRefGoogle Scholar
  9. Kamynina E, Debonneville C, Hirt RP, Staub O (2001) Liddle’s syndrome: a novel mouse Nedd4 isoform regulates the activity of the epithelial Na(+) channel. Kidney Int 60:466–471PubMedCrossRefGoogle Scholar
  10. Karpushev AV, Ilatovskaya DV, Pavlov TS, Negulyaev YA, Staruschenko A (2010) Intact cytoskeleton is required for small G protein dependent activation of the epithelial Na+ channel. PLoS One 5:e8827PubMedCrossRefGoogle Scholar
  11. Korbmacher C, Barnstable CJ (1993) Renal epithelial cells show nonselective cation channel activity and express a gene related to the cGMP-gated photoreceptor channel. EXS 66:147–164PubMedGoogle Scholar
  12. Kortenoeven ML, van den Brand M, Wetzels JF, Deen PM (2011) Hypotonicity-induced reduction of aquaporin-2 transcription in mpkCCD cells is independent of the tonicity responsive element, vasopressin, and cAMP. J Biol Chem 286:13002–13010PubMedCrossRefGoogle Scholar
  13. Lesniewska E, Giocondi MC, Vie V, Finot E, Goudonnet JP, Le Grimellec C (1998) Atomic force microscopy of renal cells: limits and prospects. Kidney Int Suppl 65:S42–S48PubMedGoogle Scholar
  14. Liu X, Yang X, Zhang B, Zhang X, Lu H, Zhang J, Zhang Y (2011) High-resolution morphological identification and characterization of living neuroblastoma SK-N-SH cells by hopping probe ion conductance microscopy. Brain Res 1386:35–40PubMedCrossRefGoogle Scholar
  15. Nascimento JM, Franchi GC Jr, Nowill AE, Collares-Buzato CB, Hyslop S (2007) Cytoskeletal rearrangement and cell death induced by Bothrops alternatus snake venom in cultured Madin–Darby canine kidney cells. Biochem Cell Biol 85:591–605PubMedCrossRefGoogle Scholar
  16. Novak P, Li C, Shevchuk AI, Stepanyan R, Caldwell M, Hughes S, Smart TG, Gorelik J, Ostanin VP, Lab MJ, Moss GWJ, Frolenkov GI, Klenerman D, Korchev YE (2009) Nanoscale live-cell imaging using hopping probe ion conductance microscopy. Nat Methods 6:279–281PubMedCrossRefGoogle Scholar
  17. Pratt JH (2005) Central role for ENaC in development of hypertension. J Am Soc Nephrol 16:3154–3159PubMedCrossRefGoogle Scholar
  18. Rheinlaender J, Geisse NA, Proksch R, Schäffer TE (2011) Comparison of scanning ion conductance microscopy with atomic force microscopy for cell imaging. Langmuir 27:697–704PubMedCrossRefGoogle Scholar
  19. Rosenthal R, Milatz S, Krug SM, Oelrich B, Schulzke JD, Amasheh S, Gunzel D, Fromm M (2010) Claudin-2, a component of the tight junction, forms a paracellular water channel. J Cell Sci 123(pt 11):1913–1921PubMedCrossRefGoogle Scholar
  20. Schild L (2010) The epithelial sodium channel and the control of sodium balance. Biochim Biophys Acta 1802:1159–1165PubMedCrossRefGoogle Scholar
  21. Shane MA, Nofziger C, Blazer-Yost BL (2006) Hormonal regulation of the epithelial Na+ channel: from amphibians to mammals. Gen Comp Endocrinol 147:85–92PubMedCrossRefGoogle Scholar
  22. Staruschenko A, Pochynyuk O, Vandewalle A, Bugaj V, Stockand JD (2007) Acute regulation of the epithelial Na+ channel by phosphatidylinositide 3-OH kinase signaling in native collecting duct principal cells. J Am Soc Nephrol 18:1652–1661PubMedCrossRefGoogle Scholar
  23. Summa V, Mordasini D, Roger F, Bens M, Martin PY, Vandewalle A, Verrey F, Feraille E (2001) Short term effect of aldosterone on Na, K-ATPase cell surface expression in kidney collecting duct cells. J Biol Chem 276:47087–47093PubMedCrossRefGoogle Scholar
  24. Takemoto F, Cohen HT, Satoh T, Katz AI (1992) Dopamine inhibits Na/K-ATPase in single tubules and cultured cells from distal nephron. Pflugers Arch 421:302–306PubMedCrossRefGoogle Scholar
  25. Yang X, Liu X, Zhang X, Lu H, Zhang J, Zhang Y (2011) Investigation of morphological and functional changes during neuronal differentiation of PC12 cells by combined hopping probe ion conductance microscopy and patch-clamp technique. Ultramicroscopy 111:1417–1422PubMedCrossRefGoogle Scholar
  26. Yang X, Liu X, Lu H, Zhang X, Ma L, Gao R, Zhang Y (2012) Real-time investigation of acute toxicity of ZnO nanoparticles on human lung epithelia with hopping probe ion conductance microscopy. Chem Res Toxicol 25:297–304PubMedCrossRefGoogle Scholar
  27. Zhang Y, Gorelik J, Sanchez D, Shevchuk A, Lab M, Vodyanoy I, Klenerman D, Edwards C, Korchev Y (2005) Scanning ion conductance microscopy reveals how a functional renal epithelial monolayer maintains its integrity. Kidney Int 68:1071–1077PubMedCrossRefGoogle Scholar
  28. Zhang Y, Sanchez D, Gorelik J, Klenerman D, Lab M, Edwards C, Korchev Y (2007) Basolateral P2X4-like receptors regulate the extracellular ATP-stimulated epithelial Na+ channel activity in renal epithelia. Am J Physiol Renal Physiol 292:1734–1740CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Occupational HygieneInstitute of Health and Environmental MedicineTianjinChina
  2. 2.Department of NeurosurgeryTianjin Medical University General Hospital, Tianjin Neurological Institute, Key Laboratory of Posttrauma Neurorepair and Regeneration in Central Nervous System, Ministry of Education, Tianjin Key Laboratory of Injuries, Variations and Regeneration of Nervous SystemTianjinChina
  3. 3.Nanomedicine LaboratoryChina National Academy of Nanotechnology and EngineeringTianjinChina

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